Development of DDPCR blood-based diagnostic tests that simultaneously measure mRNA expression from immune and cancer cells.

Abstract

22 Background: Therapeutic options for patients with non-small cell lung cancer (NSCLC) continue to expand with the advent of immunotherapies. Lack of tissue and drawbacks with available IHC tests have increased the need for blood-based diagnostics. Thus, the detection of circulating nucleic acids has become highly relevant to clinical testing. Methods: We focused on extending the utility of blood-based testing for measurement of intra-cellular transcripts to multiplexed detection of gene expression. Specifically, we addressed maximizing the yield of quality circulating RNA for use in multiplexed droplet digital PCR (ddPCR) assays. Evaluation criteria included droplet counts for biomarkers of cancer and immunotherapy response. The markers evaluated were CD45, CD3, CK8, CK18, CK19, and PD-L1. Specimens included cell lines and prospectively collected samples from normal, healthy donors and donors with NSCLC. Results: Cell lines expressing variable levels of cytokeratins and PD-L1 were used to establish assay sensitivity. In these experiments, the test system could detect these markers in the equivalent of a single cell. We evaluated specificity using RNA from these same cell lines, resting and activated lymphocytes, and monocytes. With the exception of CK8, all assays demonstrated the expected specificities. Given the complexity of assessing PD-L1 in circulation because of its expression on immune cells, a threshold of 30 copies of PD-L1 was established using normal healthy donors (n = 9). Using this cut-off we then measured PD-L1 in circulating RNA from donors with NSCLC (n = 20). By these criteria, PD-L1 expression of sufficient copy number was restricted to a single EGFR wild-type donor (1/10). Previous reports have indicated that for EGFR wild-type patients, PD-L1 over expression may be considered a poor prognostic indicator of OS. Conclusions: We are developing sensitive and specific methods that can be applied to gene expression studies in blood. We have shown feasibility of these methods by evaluating key immune and cancer-specific RNAs. Evaluations are on-going with prospective sample collections to validate thresholds for this assay that may lead to its clinical utility.